Intrauterine infection and inflammation are known risk factors for brain damage in the neonate irrespective of the gestational age. Infection‐induced maternal immune activation leads to a fetal ...inflammatory response mediated by cytokines that has been implicated in the development of not only periventricular leukomalacia and cerebral palsy but also a spectrum of neurodevelopmental disorders such as autism and schizophrenia (Behav Brain Res 2009; 204:313, Ann Neurol 2005; 57:67, Am J Obstet Gynecol 2000; 182:675). A common link among the neurobehavioral disorders associated with intrauterine inflammation appears to be the evidence for immune dysregulation in the developing brain (Behav Brain Res 2009; 204:313). The timing of the immune challenge with respect to the gestational age and neurologic development of the fetus may be crucial in the elicited response (J Neurosci 2006; 26:4752). Studies involving animal models of maternal inflammation serve a key role in elucidation of mechanisms involved in fetal brain injury associated with exposure to the maternal milieu. These animal models have been shown to result in fetal microglial activation, neurotoxicity as well motor deficits and behavioral abnormalities in the offspring (J Neurosci 2006; 26:4752, J Neurosci Res 2010; 88:172, Am J Obstet Gynecol 2009; 201:279, Am J Obstet Gynecol 2008; 199:651). A better understanding of the mechanisms of perinatal brain injury will allow discoveries of novel neuroprotective agents, better outcomes following preterm birth and stratification of fetuses and neonates for therapies in cases of preterm birth, preterm premature rupture of membranes, and chorioamnionitis.
Mitochondrial oxidative stress is associated with many neurodegenerative diseases, such as traumatic brain injury (TBI). Targeted delivery of antioxidants to mitochondria has failed to translate into ...clinical success due to their nonspecific cellular localization, poor transport properties across multiple biological barriers, and associated side effects. These challenges, coupled with the complex function of the mitochondria, create the need for innovative delivery strategies.
Neutral hydroxyl-terminated polyamidoamine (PAMAM) dendrimers have shown significant potential as nanocarriers in multiple brain injury models.
-acetyl cysteine (NAC) is a clinically used antioxidant and anti-inflammatory agent which has shown significant potency when delivered in a targeted manner. Here we present a mitochondrial targeting hydroxyl PAMAM dendrimer-drug construct (TPP-D-NAC) with triphenyl-phosphonium (TPP) for mitochondrial targeting and NAC for targeted delivery to mitochondria in injured glia. Co-localization and mitochondrial content of mitochondria-targeted and unmodified dendrimer were assessed in microglia and macrophages
via immunohistochemistry and fluorescence quantification. Therapeutic improvements of TPP-D-NAC over dendrimer-NAC conjugate (D-NAC) and free NAC were evaluated
in microglia under oxidative stress challenge.
neuroinflammation targeting was confirmed in a rabbit model of TBI.
TPP-conjugated dendrimer co-localized significantly more with mitochondria than unmodified dendrimer without altering overall levels of cellular internalization. This targeting capability translated to significant improvements in the attenuation of oxidative stress by TPP-D-NAC compared to D-NAC and free NAC. Upon systemic administration in a rabbit TBI model, TPP-conjugated dendrimer co-localized specifically with mitochondria in activated microglia and macrophages in the white matter of the ipsilateral/injured hemisphere, confirming its BBB penetration and glial targeting capabilities.
D-NAC has shown promising efficacy in many animal models of neurodegeneration, and this work provides evidence that modification for mitochondrial targeting can further enhance its therapeutic efficacy, particularly in diseases where oxidative stress-induced glial cell death plays a significant role in disease progression.
Abstract Dendrimers are an emerging group of nanostructured, polymeric biomaterials that have potential as non-viral vehicles for delivering drugs and genetic material to intracellular targets. They ...have a high charge density with tunable surface functional groups, which can alter the local environment and influence cellular interactions. This can have a significant impact on the intracellular trafficking of dendrimer-based nanodevices. With the help of flow cytometry, fluorescence microscopy, and by using specific inhibitors, the influence of surface functionality on their uptake in A549 lung epithelial cells, and subsequent intracellular distribution was investigated. In this paper, we have shown that even though all the dendrimers are taken up by fluid-phase endocytosis, significant differences in uptake mechanisms exist. Anionic dendrimers appear to be mainly taken up by caveolae mediated endocytosis in A549 lung epithelial cells, while cationic and neutral dendrimers appear to be taken in by a non-clathrin, non-caveolae mediated mechanism that may be by electrostatic interactions or other non-specific fluid-phase endocytosis. These findings open up new possibilities of targeting therapeutic agents to specific cell organelles based on surface charge.
Rett syndrome (RTT) is a pervasive developmental disorder that is progressive and has no effective cure. Immune dysregulation, oxidative stress, and excess glutamate in the brain mediated by glial ...dysfunction have been implicated in the pathogenesis and worsening of symptoms of RTT. In this study, we investigated a new nanotherapeutic approach to target glia for attenuation of brain inflammation/injury both in vitro and in vivo using a Mecp2-null mouse model of Rett syndrome.
To determine whether inflammation and immune dysregulation were potential targets for dendrimer-based therapeutics in RTT, we assessed the immune response of primary glial cells from Mecp2-null and wild-type (WT) mice to LPS. Using dendrimers that intrinsically target activated microglia and astrocytes, we studied N-acetyl cysteine (NAC) and dendrimer-conjugated N-acetyl cysteine (D-NAC) effects on inflammatory cytokines by PCR and multiplex assay in WT vs Mecp2-null glia. Since the cysteine-glutamate antiporter (Xc
) is upregulated in Mecp2-null glia when compared to WT, the role of Xc
in the uptake of NAC and L-cysteine into the cell was compared to that of D-NAC using BV2 cells in vitro. We then assessed the ability of D-NAC given systemically twice weekly to Mecp2-null mice to improve behavioral phenotype and lifespan.
We demonstrated that the mixed glia derived from Mecp2-null mice have an exaggerated inflammatory and oxidative stress response to LPS stimulation when compared to WT glia. Expression of Xc
was significantly upregulated in the Mecp2-null glia when compared to WT and was further increased in the presence of LPS stimulation. Unlike NAC, D-NAC bypasses the Xc
for cell uptake, increasing intracellular GSH levels while preventing extracellular glutamate release and excitotoxicity. Systemically administered dendrimers were localized in microglia in Mecp2-null mice, but not in age-matched WT littermates. Treatment with D-NAC significantly improved behavioral outcomes in Mecp2-null mice, but not survival.
These results suggest that delivery of drugs using dendrimer nanodevices offers a potential strategy for targeting glia and modulating oxidative stress and immune responses in RTT.
Treatment of brain injury following circulatory arrest is a challenging health issue with no viable therapeutic options. Based on studies in a clinically relevant large animal (canine) model of ...hypothermic circulatory arrest (HCA)-induced brain injury, neuroinflammation and excitotoxicity have been identified as key players in mediating the brain injury after HCA. Therapy with large doses of valproic acid (VPA) showed some neuroprotection but was associated with adverse side effects. For the first time in a large animal model, we explored whether systemically administered polyamidoamine (PAMAM) dendrimers could be effective in reaching target cells in the brain and deliver therapeutics. We showed that, upon systemic administration, hydroxyl-terminated PAMAM dendrimers are taken up in the brain of injured animals and selectively localize in the injured neurons and microglia in the brain. The biodistribution in other major organs was similar to that seen in small animal models. We studied systemic dendrimer–drug combination therapy with two clinically approved drugs, N-acetyl cysteine (NAC) (attenuating neuroinflammation) and valproic acid (attenuating excitotoxicity), building on positive outcomes in a rabbit model of perinatal brain injury. We prepared and characterized dendrimer-NAC (D-NAC) and dendrimer-VPA (D-VPA) conjugates in multigram quantities. A glutathione-sensitive linker to enable for fast intracellular release. In preliminary efficacy studies, combination therapy with D-NAC and D-VPA showed promise in this large animal model, producing 24 h neurological deficit score improvements comparable to high dose combination therapy with VPA and NAC, or free VPA, but at one-tenth the dose, while significantly reducing the adverse side effects. Since adverse side effects of drugs are exaggerated in HCA, the reduced side effects with dendrimer conjugates and suggestions of neuroprotection offer promise for these nanoscale drug delivery systems.
Emerging nanomedicine approaches in obstetrics Kannan, Rangaramanujam M., PhD; Kannan, Sujatha, MD
American journal of obstetrics and gynecology,
03/2017, Volume:
216, Issue:
3
Journal Article
Dendrimers have emerged as topical microbicides to treat vaginal infections. This study explores the
in vitro,
in vivo antimicrobial activity of PAMAM dendrimers, and the associated mechanism. ...Interestingly, topical cervical application of 500
μg of generation-4 neutral dendrimer (G
4-PAMAM-OH) showed potential to treat the
Escherichia coli induced ascending uterine infection in guinea pig model of chorioamnionitis. Amniotic fluid collected from different gestational sacs of infected guinea pigs posttreatment showed absence of
E. coli growth in the cultures plated with it. The cytokine level tumor necrosis factor (TNFα) and interleukin (IL-6 and IL-1β) in placenta of the G
4-PAMAM-OH treated animals were comparable to those in healthy animals while these were notably high in infected animals. Since, antibacterial activity of amine-terminated PAMAM dendrimers is known, the activity of hydroxyl and carboxylic acid terminated PAMAM dendrimers was compared with it. Though the G
4-PAMAM-NH
2 shows superior antibacterial activity, it was found to be cytotoxic to human cervical epithelial cell line above 10
μg/mL, while the G
4-PAMAM-OH was non-cytotoxic up to 1
mg/mL concentration. Cell integrity, outer (OM) and inner (IM) membrane permeabilization assays showed that G
4-PAMAM-OH dendrimer efficiently changed the OM permeability, while G
4-PAMAM-NH
2 and G
3.5-PAMAM-COOH damaged both OM and IM causing the bacterial lysis. The possible antibacterial mechanism are G
4-PAMAM-NH
2 acts as polycation binding to the polyanionic lipopolysaccharide in
E. coli, the G
4-PAMAM-OH forms hydrogen bonds with the hydrophilic O-antigens in
E. coli membrane and the G
3.5-PAMAM-COOH acts as a polyanion, chelating the divalent ions in outer cell membrane of
E. coli. This is the first study which shows that G
4-PAMAM-OH dendrimer acts as an antibacterial agent.
Preterm birth is a major risk factor for adverse neurological outcomes in ex-preterm children, including motor, cognitive, and behavioral disabilities. N-acetyl-L-cysteine therapy has been used in ...clinical studies; however, it requires doses that cause significant side effects. In this study, we explore the effect of low dose N-acetyl-L-cysteine therapy, delivered using a targeted, systemic, maternal, dendrimer nanoparticle (DNAC), in a mouse model of intrauterine inflammation. Our results demonstrated that intraperitoneal maternal DNAC administration significantly reduced the preterm birth rate and altered placental immune profile with decreased CD8
T-cell infiltration. Furthermore, we demonstrated that DNAC improved neurobehavioral outcomes and reduced fetal neuroinflammation and long-term microglial activation in offspring. Our study is the first to provide evidence for the role of CD8
T-cell in the maternal-fetal interface during inflammation and further support the efficacy of DNAC in preventing preterm birth and prematurity-related outcomes.
Maternal infection is a risk factor for periventricular leukomalacia and cerebral palsy (CP) in neonates. We have previously demonstrated hypomyelination and motor deficits in newborn rabbits, as ...seen in patients with cerebral palsy, following maternal intrauterine endotoxin administration. This was associated with increased microglial activation, primarily involving the periventricular region (PVR). In this study we hypothesized that maternal intrauterine inflammation leads to a pro-inflammatory environment in the PVR that is associated with microglial activation in the first 2 postnatal weeks.
Timed pregnant New Zealand white rabbits underwent laparotomy on gestational day 28 (G28). They were randomly divided to receive lipopolysaccharide (LPS; 20μg/kg in 1mL saline) (Endotoxin group) or saline (1mL) (control saline, CS group), administrated along the wall of the uterus. The PVR from the CS and Endotoxin kits were harvested at G29 (1day post-injury), postnatal day1 (PND1, 3day post-injury) and PND5 (7days post-injury) for real-time PCR, ELISA and immunohistochemistry. Kits from CS and Endotoxin groups underwent longitudinal MicroPET imaging, with 11CPK11195, a tracer for microglial activation.
We found that intrauterine endotoxin exposure resulted in pro-inflammatory microglial activation in the PVR of rabbits in the first postnatal week. This was evidenced by increased TSPO (translocator protein) expression co-localized with microglia/macrophages in the PVR, and changes in the microglial morphology (ameboid soma and retracted processes). In addition, CD11b level significantly increased with a concomitant decline in the CD45 level in the PVR at G29 and PND1. There was a significant elevation of pro-inflammatory cytokines and iNOS, and decreased anti-inflammatory markers in the Endotoxin kits at G29, PND1 and PND5. Increased 11CPK11195 binding to the TSPO measured in vivo by PET imaging in the brain of Endotoxin kits was present up to PND14–17.
Our results indicate that a robust pro-inflammatory microglial phenotype/brain milieu commenced within 24h after LPS exposure and persisted through PND5 and in vivo TSPO binding was found at PND14–17. This suggests that there may be a window of opportunity to treat after birth. Therapies aimed at inducing an anti-inflammatory phenotype in microglia might promote recovery in maternal inflammation induced neonatal brain injury.
•Maternal inflammation leads to pro-inflammatory microglial phenotype in the fetal and neonatal brain.•11C PK11195, an in vivo imaging biomarker, is upregulated in the postnatal period indicating ongoing microglial activation.•The pro-inflammatory environment provides a longer window of treatment opportunity in the postnatal period.
Polyamidoamine (PAMAM) dendrimers are multifunctional nanoparticles with tunable physicochemical features, making them promising candidates for targeted drug delivery in the central nervous system ...(CNS). Systemically administered dendrimers have been shown to localize in activated glial cells, which mediate neuroinflammation in the CNS. These dendrimers delivered drugs specifically to activated microglia, producing significant neurological improvements in multiple brain injury models, including in a neonatal rabbit model of cerebral palsy. To gain further insight into the mechanism of dendrimer cell uptake, we utilized an in vitro model of primary glial cells isolated from newborn rabbits to assess the differences in hydroxyl-terminated generation 4 PAMAM dendrimer (D4-OH) uptake by activated and non-activated glial cells. We used fluorescently-labelled D4-OH (D-Cy5) as a tool for investigating the mechanism of dendrimer uptake. D4-OH PAMAM dendrimer uptake was determined by fluorescence quantification using confocal microscopy and flow cytometry. Our results indicate that although microglial cells in the mixed cell population demonstrate early uptake of dendrimers in this in vitro system, activated microglia take up more dendrimer compared to resting microglia. Astrocytes showed delayed and limited uptake. We also illustrated the differences in mechanism of uptake between resting and activated microglia using different pathway inhibitors. Both resting and activated microglia primarily employed endocytotic pathways, which are enhanced in activated microglial cells. Additionally, we demonstrated that hydroxyl terminated dendrimers are taken up by primary microglia using other mechanisms including pinocytosis, caveolae, and aquaporin channels for dendrimer uptake.
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